人抗凝血酶Ⅲ的cDNA克
發(fā)布時(shí)間：2019-05-28 07:46
【摘要】：與傳統(tǒng)的藥物相比,重組蛋白質(zhì)藥物具有活性高、特異性強(qiáng)、毒性低、生物功能明確、便于臨床應(yīng)用、可規(guī)模化生產(chǎn)的優(yōu)點(diǎn),因此,重組藥物蛋白的生產(chǎn)是當(dāng)今國(guó)內(nèi)外藥物研究的熱點(diǎn),其中研究的重點(diǎn)則是如何經(jīng)濟(jì)、高效的生產(chǎn)和應(yīng)用這類蛋白。雖然有細(xì)菌、真菌、昆蟲(chóng)細(xì)胞、動(dòng)物細(xì)胞、轉(zhuǎn)基因動(dòng)植物等幾種外源蛋白表達(dá)系統(tǒng)用來(lái)生產(chǎn)重組藥用蛋白,但它們均存在著一些難以彌補(bǔ)的缺陷。為探討利用動(dòng)物乳腺作為表達(dá)系統(tǒng)來(lái)生產(chǎn)藥用蛋白的可行性,本研究選取人血漿中重要的抗凝蛋白-人抗凝血酶(hAT)作為研究對(duì)象,構(gòu)建含有hAT cDNA基因的重組腺病毒載體Ad-hAT,將Ad-hAT注入羊乳腺內(nèi),感染羊乳腺上皮細(xì)胞,使hAT的cDNA基因在羊乳腺內(nèi)高效表達(dá),從而在羊乳汁中獲得具有生物功能的重組人抗凝血酶(rhAT)。對(duì)rhAT進(jìn)行分離純化后,作用于彌散性靜脈內(nèi)凝血(DIC)大鼠模型,對(duì)rhAT治療DIC大鼠的療效進(jìn)行評(píng)判。通過(guò)以上試驗(yàn),為重組腺病毒載體介導(dǎo)人源基因在動(dòng)物乳腺中高效表達(dá)rhAT以及rhAT在人類DIC疾病中的應(yīng)用提供理論依據(jù)和技術(shù)支持。 本研究主要包括以下內(nèi)容:(1)提取人胚胎肝臟組織中的總RNA,經(jīng)RT-PCR,獲得用于表達(dá)的hAT的cDNA基因序列。對(duì)獲得的cDNA序列進(jìn)行測(cè)序分析,初步確定所擴(kuò)增的cDNA的正確性。(2)以pcDNA3.1(+)為基礎(chǔ)質(zhì)粒,構(gòu)建重組真核表達(dá)質(zhì)粒p3AT,將之轉(zhuǎn)入體外培養(yǎng)的羊乳腺上皮細(xì)胞細(xì)胞中獲得表達(dá),進(jìn)一步證實(shí)hAT的cDNA序列可用于表達(dá)rhAT;(3)以pShuttle-CMV為基礎(chǔ)質(zhì)粒,構(gòu)建重組穿梭載體pShAT,細(xì)菌內(nèi)同源重組法獲得重組腺病毒載體質(zhì)粒pAd-hAT;(4)將pAd-hAT轉(zhuǎn)入HEK293細(xì)胞中,通過(guò)病毒包裝獲得重組hAT腺病毒載體Ad-hAT;Ad-hAT在HEK293細(xì)胞內(nèi)大量擴(kuò)增;(5)將Ad-hAT轉(zhuǎn)入體外培養(yǎng)的羊乳腺上皮細(xì)胞中獲得表達(dá),進(jìn)一步證實(shí)Ad-hAT可用于高效表達(dá)rhAT;(6)將Ad-hAT注入羊乳腺,感染乳腺上皮細(xì)胞,在乳汁中獲得rhAT,對(duì)乳汁中rhAT進(jìn)行檢測(cè);應(yīng)用肝素瓊脂糖凝膠6FF親和層析法分離乳汁中的重組蛋白,獲得rhAT凍干粉;(7)構(gòu)建大鼠DIC動(dòng)物模型,分別用人血漿提取的hAT(hpAT)和rhAT進(jìn)行處理,檢測(cè)DIC大鼠血漿中DIC指標(biāo),進(jìn)行療效判定;(8)對(duì)hpAT和rhAT對(duì)DIC大鼠的療效進(jìn)行比較,探索在DIC治療中,利用rhAT取代hpAT的可行性。 進(jìn)行上述試驗(yàn)研究,取得如下研究結(jié)果: (1)通過(guò)RT-PCR法從人胚胎肝臟組織中獲得了hAT的cDNA基因。經(jīng)DNA測(cè)序分析,所得hAT的cDNA與Genbank中的相應(yīng)序列(NM_000488)一致。 (2)以pcDNA3.1+、pIRES和pShuttle-CMV為基礎(chǔ)質(zhì)粒,構(gòu)建了含有hAT cDNA基因的真核表達(dá)載體p3AT以及穿梭表達(dá)載體pShAT。 載體p3AT含有新霉素抗性篩選標(biāo)記基因和GFP報(bào)告基因,可對(duì)已轉(zhuǎn)染的哺乳動(dòng)物細(xì)胞進(jìn)行抗性篩選,也可對(duì)hAT的表達(dá)進(jìn)行實(shí)時(shí)觀測(cè);構(gòu)建的pShAT質(zhì)粒含有GFP報(bào)告基因,可對(duì)腺病毒載體進(jìn)行實(shí)時(shí)蛋白表達(dá)觀測(cè)和快速滴度測(cè)定,為下一步研究rhAT的表達(dá)奠定基礎(chǔ)。 (3)將所構(gòu)建并經(jīng)鑒定正確的pShAT用Pme I酶切線性化,將所獲得的線性化片斷直接轉(zhuǎn)化處于感受態(tài)且含有Adeasy-1質(zhì)粒的大腸桿菌BJ5183,通過(guò)細(xì)菌內(nèi)同源重組的方法獲得了含有hAT cDNA基因的重組腺病毒載體質(zhì)粒pAd-hAT。證實(shí)細(xì)菌內(nèi)同源重組能高效獲得重組腺病毒載體質(zhì)粒。 (4)在培養(yǎng)的HEK293細(xì)胞中,進(jìn)行了重組腺病毒Ad-hAT的包裝與擴(kuò)增,獲得了重組腺病毒Ad-hAT。 (5)質(zhì)粒表達(dá)載體p3AT在體外培養(yǎng)的羊乳腺上皮細(xì)胞中獲得表達(dá),表達(dá)含量達(dá)到了700±90 mg/L,rhAT活性為110%～120%,證實(shí)了所PT-PCR獲得的hAT cDNA可用于hAT的表達(dá);重組腺病毒載體Ad-hAT在體外培養(yǎng)的羊乳腺上皮細(xì)胞中獲得表達(dá),表達(dá)含量達(dá)到了900±85 mg/L,rhAT活性為120%～130%,證實(shí)了Ad-hAT在羊乳腺上皮細(xì)胞中表達(dá)的可能性,為下一步在羊乳腺內(nèi)高效表達(dá)hAT奠定了基礎(chǔ)。 (6)將Ad-hAT注入羊乳腺后,在乳汁中獲得了rhAT的高效表達(dá),21 d內(nèi)共11次的檢測(cè)平均表達(dá)含量達(dá)到1.58±0.21 g/L,日檢測(cè)單次最高3.1 g/L,活性平均為102.5±1.44 %。應(yīng)用肝素瓊脂糖凝膠親和層析法分離純化rhAT后,分離純化后的rhAT回收率達(dá)到54.7±1.0%,純度達(dá)到92.5±0.5%。 (7)成功構(gòu)建大鼠DIC動(dòng)物模型。 (8)用rhAT和hpAT處理DIC大鼠后,rhAT和hpAT一樣(P0.05),均有效地緩解了大鼠DIC病情,對(duì)治療DIC大鼠具有良好的療效,主要表現(xiàn)為:血漿纖維蛋白原濃度減少和血小板增多的趨勢(shì)得到有效抑制;血漿內(nèi)hAT活性和凝血酶-抗凝血酶復(fù)合物(TAT)濃度得到相應(yīng)增加;谷丙轉(zhuǎn)氨酶(ALT)和谷草轉(zhuǎn)氨酶(AST)濃度上升的趨勢(shì)得到有效抑制。結(jié)果說(shuō)明,在DIC的治療藥物選擇中,rhAT具有取代hpAT的可行性。 本研究利用腺病毒為載體,在羊乳腺中高效表達(dá)了具有生物活性的rhAT;應(yīng)用肝素瓊脂糖凝膠親和層析法分離純化rhAT,獲得了較高純度的rhAT凍干粉;應(yīng)用rhAT作用DIC大鼠模型,取得了和hpAT一樣的良好的治療效果(P0.05)。研究結(jié)果充分說(shuō)明,本試驗(yàn)確定的重組蛋白表達(dá)與分離純化路線是切實(shí)可行的,該方法獲得的rhAT在治療DIC疾病方面具有和hpAT同等的療效。這些,將促進(jìn)從人血漿中提取hAT這一傳統(tǒng)生產(chǎn)方式的變革,使hAT生產(chǎn)向高效、經(jīng)濟(jì)、規(guī)�；较虬l(fā)展;也將進(jìn)一步拓寬rhAT的應(yīng)用領(lǐng)域,創(chuàng)造出更大的經(jīng)濟(jì)和社會(huì)效益。
[Abstract]:Compared with the traditional medicine, the recombinant protein medicine has the advantages of high activity, strong specificity, low toxicity, definite biological function, convenient clinical application and large-scale production, The focus of the study was on how to produce and apply such proteins economically and efficiently. Although several foreign protein expression systems, such as bacteria, fungi, insect cells, animal cells, transgenic animals and plants, are used to produce recombinant medicinal proteins, they all have a number of defects that are difficult to compensate. In order to study the feasibility of using animal mammary gland as the expression system to produce the medicinal protein, the important anti-coagulation protein-human anti-thrombin (hAT) in human plasma was used as the research object, and the recombinant adenovirus vector Ad-hAT containing the hAT cDNA gene was constructed, and Ad-hAT was injected into the mammary gland of the sheep. The sheep mammary epithelial cells are infected, and the cDNA gene of the hAT is efficiently expressed in the mammary gland of the sheep, so as to obtain the recombinant human anti-thrombin (rhAT) with the biological function in the milk of the sheep. After the separation and purification of rhAT, the model of disseminated intravascular coagulation (DIC) was applied to evaluate the efficacy of rhAT in the treatment of DIC rats. Through the above test, the recombinant adenovirus vector-mediated human gene is used for efficiently expressing rhAT in the mammary gland of the animal and the application of the rhAT in the human DIC disease is provided with the theoretical foundation and the technical support. The main contents of this study are as follows: (1) The total RNA in human embryonic liver tissue is extracted, and the cDNA gene of hAT for expression is obtained through RT-PCR. and sequencing and analyzing the obtained cDNA sequence to preliminarily determine the positive and negative of the amplified cDNA, and (2) constructing a recombinant eukaryotic expression plasmid p3AT by using the pcDNA3.1 (+) as a base plasmid, transferring the recombinant eukaryotic expression plasmid p3AT into an in-vitro cultured goat mammary epithelial cell cell to obtain the expression, further confirming that the cDNA sequence of the hAT can be used for expressing the rhAT, (3) constructing a recombinant shuttle vector pSh with the pShuttle-CMV as a base plasmid, in that method, the recombinant adenovirus vector plasmid pAd-hAT is obtained by the homologous recombination in the AT and the bacteria; (4) the pAd-hAT is transferred into the HEK293 cell, and the recombinant hAT adenovirus vector Ad-hAT is obtained through the virus package; and the Ad-hAT is amplified in a large amount in the HEK293 cell; and (5) the Ad-hAT is transferred into the goat mammary epithelial cell cultured in vitro the expression of the Ad-hAT can be further confirmed to be used for high-efficiency expression of rhAT; (6) the Ad-hAT is injected into the mammary gland of the sheep, the human mammary epithelial cell is infected, rhAT is obtained in the milk, the rhAT in the milk is detected, the recombinant protein in the milk is separated by the heparin agarose gel 6-FF affinity chromatography, and the rhAT is obtained The effects of hpAT and rhAT on DIC rats were compared, and the effect of hpAT and rhAT on DIC rats was compared, and in the treatment of DIC, it was found that rhAT could be used to replace hpAT. 1. Carry out the above-mentioned test and study, and obtain the The results of the following studies: (1) hA was obtained from human fetal liver tissue by RT-PCR The cDNA of T. The cDNA of hAT and the corresponding sequence in Genbank (NM _ 0) were analyzed by DNA sequencing. (2) Eukaryotic expression vector p3AT containing hAT cDNA gene was constructed with pcDNA3.1 +, pIRES and pShuttle-CMV. The shuttle expression vector pSHAT. The vector p3AT contains a neomycin resistance screening marker gene and a GFP reporter gene, and the transfected mammalian cells can be screened for resistance, and the expression of the hAT can be observed in real time; and the constructed pS The hAT plasmid contains the GFP reporter gene, and can carry out real-time protein expression observation and rapid titer determination on the adenovirus vector, And the obtained linearized fragment is directly transformed into a competent state and contains the Adeasy-1 plasmid, and the obtained linearized fragment is directly transformed into the E. coli BJ5183 which is competent and contains the Adeasy-1 plasmid, and the hAT cDNA gene is obtained through the method of homologous recombination in the bacterium. The recombinant adenovirus vector pAd-hAT. (4) The recombinant adenovirus Ad-hAT was carried out in the cultured HEK293 cells. The expression of the recombinant adenovirus Ad-hAT. (5) plasmid expression vector p3AT was obtained in the goat mammary epithelial cells cultured in vitro, the expression level of the recombinant adenovirus Ad-hAT was 700-90 mg/ L, the activity of rhAT was 110-120%, and the PT was confirmed. the hAT cDNA obtained by-PCR can be used for the expression of hAT; the recombinant adenovirus vector Ad-hAT is expressed in the goat mammary epithelial cell cultured in vitro, the expression content of the recombinant adenovirus vector Ad-hAT is 900 to 85mg/ L, the activity of the rhAT is 120 to 130 percent, and the possibility of the expression of the Ad-hAT in the goat mammary epithelial cell is confirmed, The expression of hAT in the breast of the sheep was established by the next step. (6) After the Ad-hAT was injected into the breast of the sheep, the high-efficiency expression of rhAT was obtained in the milk, and the average expression content of 11 times in 21 days was 1.58-0.21g/ L. The highest activity was 3.1 g/ L, the average activity was 102.5 to 1.44%, and the purified rhAT was isolated and purified by a heparin-agarose gel affinity chromatography, and the purified rhAT was recovered. The rate reaches 54.7% to 1.0%, and the purity is up to 92.5 to 0.5%. (7) The rat model of DIC was successfully constructed. (8) After treatment with rhAT and hpAT, rhAT and hpAT were the same (P0.05). The main performance is that the plasma fibrinogen concentration is reduced and the tendency of the platelet increase is effectively inhibited; the concentration of the hAT activity and the thrombin-antithrombin complex (TAT) in the plasma is correspondingly increased; The increase in the concentration of transaminases (ALT) and aspartate aminotransferase (AST) was effectively inhibited. In the treatment of DIC, rhAT has the feasibility of replacing hpAT. hAT, high-purity rhAT freeze-dried powder is obtained, rhAT is applied, The results of the study show that the recombinant protein expression and the separation and purification route determined by this test are practical and feasible. The rhAT obtained by the method has the same curative effect as hpAT in the treatment of DIC diseases,
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R341
本文編號(hào)：2486870
[Abstract]:Compared with the traditional medicine, the recombinant protein medicine has the advantages of high activity, strong specificity, low toxicity, definite biological function, convenient clinical application and large-scale production, The focus of the study was on how to produce and apply such proteins economically and efficiently. Although several foreign protein expression systems, such as bacteria, fungi, insect cells, animal cells, transgenic animals and plants, are used to produce recombinant medicinal proteins, they all have a number of defects that are difficult to compensate. In order to study the feasibility of using animal mammary gland as the expression system to produce the medicinal protein, the important anti-coagulation protein-human anti-thrombin (hAT) in human plasma was used as the research object, and the recombinant adenovirus vector Ad-hAT containing the hAT cDNA gene was constructed, and Ad-hAT was injected into the mammary gland of the sheep. The sheep mammary epithelial cells are infected, and the cDNA gene of the hAT is efficiently expressed in the mammary gland of the sheep, so as to obtain the recombinant human anti-thrombin (rhAT) with the biological function in the milk of the sheep. After the separation and purification of rhAT, the model of disseminated intravascular coagulation (DIC) was applied to evaluate the efficacy of rhAT in the treatment of DIC rats. Through the above test, the recombinant adenovirus vector-mediated human gene is used for efficiently expressing rhAT in the mammary gland of the animal and the application of the rhAT in the human DIC disease is provided with the theoretical foundation and the technical support. The main contents of this study are as follows: (1) The total RNA in human embryonic liver tissue is extracted, and the cDNA gene of hAT for expression is obtained through RT-PCR. and sequencing and analyzing the obtained cDNA sequence to preliminarily determine the positive and negative of the amplified cDNA, and (2) constructing a recombinant eukaryotic expression plasmid p3AT by using the pcDNA3.1 (+) as a base plasmid, transferring the recombinant eukaryotic expression plasmid p3AT into an in-vitro cultured goat mammary epithelial cell cell to obtain the expression, further confirming that the cDNA sequence of the hAT can be used for expressing the rhAT, (3) constructing a recombinant shuttle vector pSh with the pShuttle-CMV as a base plasmid, in that method, the recombinant adenovirus vector plasmid pAd-hAT is obtained by the homologous recombination in the AT and the bacteria; (4) the pAd-hAT is transferred into the HEK293 cell, and the recombinant hAT adenovirus vector Ad-hAT is obtained through the virus package; and the Ad-hAT is amplified in a large amount in the HEK293 cell; and (5) the Ad-hAT is transferred into the goat mammary epithelial cell cultured in vitro the expression of the Ad-hAT can be further confirmed to be used for high-efficiency expression of rhAT; (6) the Ad-hAT is injected into the mammary gland of the sheep, the human mammary epithelial cell is infected, rhAT is obtained in the milk, the rhAT in the milk is detected, the recombinant protein in the milk is separated by the heparin agarose gel 6-FF affinity chromatography, and the rhAT is obtained The effects of hpAT and rhAT on DIC rats were compared, and the effect of hpAT and rhAT on DIC rats was compared, and in the treatment of DIC, it was found that rhAT could be used to replace hpAT. 1. Carry out the above-mentioned test and study, and obtain the The results of the following studies: (1) hA was obtained from human fetal liver tissue by RT-PCR The cDNA of T. The cDNA of hAT and the corresponding sequence in Genbank (NM _ 0) were analyzed by DNA sequencing. (2) Eukaryotic expression vector p3AT containing hAT cDNA gene was constructed with pcDNA3.1 +, pIRES and pShuttle-CMV. The shuttle expression vector pSHAT. The vector p3AT contains a neomycin resistance screening marker gene and a GFP reporter gene, and the transfected mammalian cells can be screened for resistance, and the expression of the hAT can be observed in real time; and the constructed pS The hAT plasmid contains the GFP reporter gene, and can carry out real-time protein expression observation and rapid titer determination on the adenovirus vector, And the obtained linearized fragment is directly transformed into a competent state and contains the Adeasy-1 plasmid, and the obtained linearized fragment is directly transformed into the E. coli BJ5183 which is competent and contains the Adeasy-1 plasmid, and the hAT cDNA gene is obtained through the method of homologous recombination in the bacterium. The recombinant adenovirus vector pAd-hAT. (4) The recombinant adenovirus Ad-hAT was carried out in the cultured HEK293 cells. The expression of the recombinant adenovirus Ad-hAT. (5) plasmid expression vector p3AT was obtained in the goat mammary epithelial cells cultured in vitro, the expression level of the recombinant adenovirus Ad-hAT was 700-90 mg/ L, the activity of rhAT was 110-120%, and the PT was confirmed. the hAT cDNA obtained by-PCR can be used for the expression of hAT; the recombinant adenovirus vector Ad-hAT is expressed in the goat mammary epithelial cell cultured in vitro, the expression content of the recombinant adenovirus vector Ad-hAT is 900 to 85mg/ L, the activity of the rhAT is 120 to 130 percent, and the possibility of the expression of the Ad-hAT in the goat mammary epithelial cell is confirmed, The expression of hAT in the breast of the sheep was established by the next step. (6) After the Ad-hAT was injected into the breast of the sheep, the high-efficiency expression of rhAT was obtained in the milk, and the average expression content of 11 times in 21 days was 1.58-0.21g/ L. The highest activity was 3.1 g/ L, the average activity was 102.5 to 1.44%, and the purified rhAT was isolated and purified by a heparin-agarose gel affinity chromatography, and the purified rhAT was recovered. The rate reaches 54.7% to 1.0%, and the purity is up to 92.5 to 0.5%. (7) The rat model of DIC was successfully constructed. (8) After treatment with rhAT and hpAT, rhAT and hpAT were the same (P0.05). The main performance is that the plasma fibrinogen concentration is reduced and the tendency of the platelet increase is effectively inhibited; the concentration of the hAT activity and the thrombin-antithrombin complex (TAT) in the plasma is correspondingly increased; The increase in the concentration of transaminases (ALT) and aspartate aminotransferase (AST) was effectively inhibited. In the treatment of DIC, rhAT has the feasibility of replacing hpAT. hAT, high-purity rhAT freeze-dried powder is obtained, rhAT is applied, The results of the study show that the recombinant protein expression and the separation and purification route determined by this test are practical and feasible. The rhAT obtained by the method has the same curative effect as hpAT in the treatment of DIC diseases,
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R341
【引證文獻(xiàn)】
相關(guān)期刊論文 前1條
1 李秋菊;王巍;馬紅霞;高云航;高見(jiàn);;抗凝血酶概述[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2011年09期
，本文編號(hào)：2486870
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